After being administered orally, most protein and peptide drugs lose their active structures in the acidic environment of the stomach or undergo enzymatic degradation. Also, they are adsorbed at very low rates through the gastric or intestinal mucosa. For these reasons, protein or peptide drugs usually take non-oral administration routes, that is, are usually administered by injection. The non-oral administration of protein or peptide drugs must be repeated because most non-orally administered protein or peptide drugs show short half lives and low bioavailability in the body. In addition, their administration may be continued for a long time period, e.g., months, in many cases. In order to avoid these problems, active research into sustained-release dosage formulations has been conducted, resulting in the use of biodegradable polymeric carriers with protein or peptide drugs encapsulated therein, which can release the protein or peptide drugs therefrom as the biodegradation of the polymeric carriers progresses [Heller, J. et al., Controlled release of water-soluble macromolecules from bioerodible hydrogels, Biomaterials, 4, 262-266, 1983; Langer, R., New methods of drug delivery, Science, 249, 1527-1533, 1990; Langer, R., Chem. Eng. Commun., 6, 1-48, 1980; Langer, R. S. and Peppas, N. A., Biomaterials, 2, 201-214, 1981; Heller, J., CRC Crit. Rev. Ther. Drug Carrier Syst., 1(1), 39-90, 1984; Holland, S. J. Tighe, B. J. and Gould, P. L., J. Controlled Release, 155-180, 1986].
Aliphatic polyesters, currently used as polymeric carriers for protein or peptide drugs, received FDA permission for the use thereof because their biocompatibility was recognized. They are widely used as carriers for drug delivery or sutures for operations. Concrete examples of aliphatic polyesters include poly-L-lactic acid, polyglycolic acid, poly-D-lactic acid-co-glycolic acid, poly-L-lactic acid-co-glycolic acid, poly-D,L-lactic acid-co-glycolic acid (hereinafter referred to as ‘PLGA’), poly-caprolactone, poly-valerolactone, poly-hydroxy butyrate and poly-hydroxy valerate [Peppas, L. B., Int. J. Pharm., 116, 1-9, 1995].
With the development of high-molecular weight peptides or proteins as novel therapeutics in recent years, various attempts have been made to release them from polymeric carriers in a controlled manner. The dosage forms comprising polyester microspheres with protein drugs encapsulated therein, however, suffer from the disadvantages of showing an initial burst effect, an uncontrolled release rate for a period of time due to various factors, or an incomplete release of the encapsulated drug.
For example, model protein drugs, such as bovine serum albumin, lysozyme, etc. are released in large amounts in an initial stage, but show a final release of around 50% [Crotts, G. and Park, T. G., J. Control. Release, 44, 123-134, 1997; Leonard, N. B., Michael, L. H., Lee, M. M. J. Pharm. Sci., 84, 707-712]. As for microspheres using aliphatic polyester carriers with recombinant human growth hormone encapsulated therein, they initially release the drug in an amount of 30-50%, but 40-60% of the drug remains in the microspheres [Yan, C., et al., J. Control. Release, 32, 231-241, 1994; Kim, H. K. and Park, T. G., Biotechnol. Bioeng., 65, 659-667, 1999].
The initial burst release of the drug is attributed to the fact that proteinous drugs aggregated at or adsorbed to microsphere surfaces or holes are released through rapid diffusion in an initial stage.
Proteinous drugs may be denatured by the interface between water and an organic solvent during the manufacture of microspheres, and thus form irreversible aggregates which lead to unstable release. In order to prevent the interface-induced denaturation of proteinous drugs, the use of surfactants (e.g., non-ionic type surfactant Tween, Pluronic F68, Brij 35, etc.) and stabilizers (e.g., mannitol, gelatin, trehalose, carboxymethylcellulose, etc.) or an organic solvent free of water in the preparation of microspheres has been reported [Gombotz, W. R., Healy, M., Brown, L., U.S. Pat. No. 5,019,400].
In order to solve the problem of uncontrollable drug release rates for a period of time and the incomplete release of encapsulated drugs, many recent studies are associated with alternative methods of preparing microspheres for the sustained release of drugs, which include encapsulating a drug in a mixture of two or more polymers with different degradation rates at a predetermined ratio [Ravivarapu, H. B., Burton, K., Deluca, P. P., Eur J Pharm Biopharm 50(2) 263-270, 2000; Korean Patent Application No. 1998-0062142] or mixing two or more polymeric microspheres having different degradation rates with respective drugs encapsulated therein at a predetermined ratio (U.S. Pat. No. 4,897,268), thereby controlling both the initial release and the continuous release of the drug or drugs from the microspheres. In the microspheres prepared by the conventional methods, however, the products degraded from the polymer with a high degradation rate, e.g., lactic acid and glycolic acid, decrease the pH value, which promotes the degradation of the polymer having a low degradation rate, resulting in a release rate quite different from the calculated means of the release rates of the drugs encapsulated in respective polymers. Further, the preparation of two or more microspheres for one dosage form is disadvantageous in terms of manufacturing processes and economy (Korean Patent Application No. 2000-0036178).
As techniques for preparing microspheres, phase separation (U.S. Pat. No. 4,673,595, Korean Pat. Application No. 2007-0031304), spray-drying (Korean Pat. Application No. 2003-0023130) and organic solvent evaporation (U.S. Pat. No. 4,389,330) are generally known. In a phase separation method, a methylene chloride solvent is used in combination with silicon oil, heptene and ethyl alcohol, but all of them have to be eliminated and thus are economically disadvantageous. As for the spray-drying method, it may incur the denaturation of a peptide or proteinous drug because it requires the spray-drying of the peptide or proteinous drug at a high temperature, such as 60° C. or higher, along with an organic solvent. For these reasons, the organic solvent evaporation method is most widely applied to the preparation of peptide or proteinous drugs. One of the most technically important factors in this method is encapsulation efficiency (Korean Patent Application No. 2003-0081179).
Therefore, there is the need for a preparation method of microspheres that shows neither an initial burst effect nor incomplete release, allows the zero-order release of drugs, is simple and economically advantageous and ensures high encapsulation efficiency and high stability of the encapsulated drug.
Glucose-regulating peptides belong to a group of peptides which have therapeutic potential for the treatment of insulin-dependent diabetes mellitus, gestational diabetes mellitus or insulin-independent diabetes mellitus, obesity and lipid dysmetabolism (U.S. Pat. No. 6,506,724). Examples of glucose-regulating peptides include Exendin-3, Exendin-4 and homologs and agonists thereof, and glucagons, glucagons-like peptides (e.g., GLP-1, GLP-2) and homologs and agonists thereof (Korean Patent Application No. 2006-7015029).
Exendin-4, isolated from the salivary secretions of the lizard Heloderma horridum or Heloderma suspectum, is a physiologically active peptide consisting of 39 amino acid residues. Exendin-4 functions to stimulate the secretion of insulin from pancreatic beta cells, reduce elevated glucagons secretion and induce a decrease of appetite, thereby being useful for the treatment of diabetes and obesity [Eng. J. et al. 1990; Raufman, J. P. 1992; Goeke, R. 1993; Thorens, B. 1993].
For the effective prevention and treatment of diabetes mellitus, studies on microspheres for the sustained release of exendin-4 have been conducted (Korean Patent Application No. 2006-7023921). However, conventional methods are complicated and inefficient, as exemplified by the use and removal of many organic solvents in the phase separation method, peptide degradation attributable to the use of high energy in an ultrasonic process, and the use of many excipients including stabilizers such as sugar, and release enhancers (e.g., inorganic acids and inorganic salts).